In recent years, gene testing has been becoming rapidly widely used in the clinical diagnosis field. With gene testing, nucleic acids and chromosomes are analyzed to allow for examination of presence or absence of variations and nuclear forms associated with hereditary disorders for clinical purposes. As one example of gene testing, diagnosis of cancer cell metastasis to lymph nodes is mentioned. Cancer cells leave primary tumor and spread by metastasis all over patient's body via blood vessels and lymph ducts. At operation of a cancer, lesions should be removed as much surely as possible, and therefore, metastasis should be detected accurately and appropriate treatments be provided depending on the degree of metastasis. In this sense, diagnosis of cancer cell metastasis to lymph nodes made during the operation has an extremely important meaning. As one of methods of diagnosis of cancer cell metastasis to lymph nodes, such a method is known that nucleic acid of a protein, that is not expressed in normal cells or expression level thereof is low but is expressed a great deal in cancer cells, is detected as a target nucleic acid. Thanks to advancement of gene analysis technology made in these years, it is now possible to perform cancer diagnosis effectively by detecting a target nucleic acid contained in the lymph node tissue resected from the living organism through amplification.
As mentioned above, when it is desired to make judgment of cancer cell metastasis to lymph nodes by amplification of target nucleic acid, amplification of target nucleic acids in a measurement sample is performed using a measurement sample which is prepared in such that lymph nodes are homogenized and target nucleic acids are extracted into a solution and purified. However, with this method, there is such a drawback that purification of the target nucleic acid needs considerable time, it takes longer time before results of the judgment by amplification of the target nucleic acid are made available, and it is difficult to perform promptly diagnosis of cancer cell metastasis by amplification of the target nucleic acid. At diagnosis of cancer cell metastasis to lymph nodes during the operation, treatment strategy in the operation is determined according to results of judgment of metastasis of cancer cells, and therefore, quick judgment of metastasis is so important.
From viewpoints mentioned above, if a solution in which lymph nodes are homogenized or supernatant of this solution is used as the measurement sample without executing extraction and purification of the nucleic acid at the time of preparation of measurement sample, it is possible to perform measurements of the target nucleic acid in prompt fashion. However, when the target nucleic acid is amplified using such a measurement sample, there is such a problem that, compared to a case where amplification of nucleic acids is made using a measurement sample prepared by purification of the nucleic acid, the amount of inhibitory substances that prevent amplification of the target nucleic acid derived from lymph nodes increases and accurate measurement results can not be thus obtained.
In order to overcome this problem, conventionally, such a method is known that amplification of a target nucleic acid is estimated using a nucleic acid probe which hybridizes to the target nucleic acid (see, for example, Japanese Patent Application Laid-Open No. 2004-203). According to the method disclosed in Japanese Patent Application Laid-Open No. 2004-203, a nucleic acid (internal standard nucleic acid) in which base sequence of the target nucleic acid is mutated in part is added to a measuring system at known concentration, and at the same time, a target nucleic acid probe that hybridizes specifically to the target nucleic acid and an internal standard nucleic acid probe that hybridizes specifically to the internal standard nucleic acid are added to the measuring system, the target nucleic acid and the internal standard nucleic acid are then measured at one time using PCR method, and the target nucleic acid is measured from an amount of addition of the internal standard nucleic acid.
However, with the method for measuring the target nucleic acid disclosed in above-mentioned Japanese Patent Application Laid-Open No. 2004-203, the internal standard nucleic acid does not necessarily exhibit the same reactivity as the target nucleic acid does, and therefore, a problem arises that accurate measurement of the target nucleic acid is difficult.